Characterization of human tumor samples and cell lines in combination with inhibitor studies in animal models has established a central role for the Hh pathway in a vast array of cancer types, including small-cell lung, pancreatic, oesophageal, prostate, breast, colon, liver and ovarian cancers. Hh signaling is now implicated in approximately 20-25% of all cancers. GLI1 is the downstream effector in the Hh signaling pathway, and has emerged as a valid therapeutic target. It has been suggested that due to the complexity of signaling inputs in the Hh pathway, targeting GLI1 may provide a more comprehensive strategy for treating both canonical and noncanonical Hh-pathway dependent cancers. This is especially true in light of the acquired resistance to inhibitors of SMO in patients. Screens utilizing GLI1-dependent transcriptional reporter cell-based assays have yielded inhibitors targeting the Hh pathway downstream of SMO including small molecule and natural product compounds that act by targeting GLI1 through differing mechanisms of action. Targeting at the level of GLI1 with small molecules has been effective in a number of cancer model systems including colon, CLL and breast cancer. However, these compounds have only micromolar potencies in vitro, and a subset (GANT61 and ATO) has shown only modest efficacy in in vivo cancer models. These studies suggest that GLI1 is pharmacologically targetable, and that the use of GLI1 inhibitors is a valid and promising approach for targeting GLI1-dependent cancers. Unfortunately, current GLI1 inhibitors are hampered by low potency and a lack of in vivo efficacy and hence are not viable clinical candidates. There are no GLI1-targeted inhibitors in the clinic. Using a novel high throughput, high content cell-based imaging platform, we will screen a diverse collection of small molecules to identify potent inhibitors of GLI1 (Aim 1). Compounds that show inhibitory activity will be further validated in a focused panel of cell-based assays which will incorporate cells derived from primary human breast tumors. These assays will also examine selectivity of the compounds as well as their effects on motility/invasiveness (Aim 2). Promising compounds discovered in Aims 1 and 2 will be subjected to extensive structure-activity relationship to optimize the novel GLI1 inhibitors (Aim 3).